1. Field of the Invention
Pharmacological research has developed—starting out from the use of exclusively natural sources, via the chemical synthesis of active substances and their testing by means of animal experiments—toward the targeted, computer-aided structure design of active substances using experimental and theoretical methods.
2. Description of the Related Art
With increased knowledge of the various causes of disease (e.g. lack or genetically caused alteration of a protein) pharmaceutical research and therapy by medicaments have become considerably more complex. Thus, over the past ten years, the genetic causes of some primarily neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prionic diseases and various ataxic syndroms could be elucidated by means of molecular-biological methods (Human Genome Project). This recognition of the biological changes underlying the diseases forms the basis for a shift from a symptomatic, palliative towards a causal therapy.
100 to 150 of the around 30.000 diseases described in medicine are relevant enough to be suitable as research projects for the pharmaceutical industry. The medicaments currently available aim at therapeutically influencing approx. 400 receptors, enzymes and other biomolecules. It is assumed, however, that approximately up to 10,000 genes and products thereof are suitable as targets for active agent research. Proving their pathological relevance requires, inter alia, molecular and cellular systems of informative value.
Apart from the rational design, which involves optimization of substance properties based on empirical values or based on known molecular structures, currently combinatorial chemistry and combinatorial biosynthesis, the latter being in the development stage, play an important part in drug research.
An important weak point of these methods is the limited diversity of synthetic substances compared to the structural complexity of vegetable and microbial secondary metabolites.
To be able to exploit this natural diversity, it is indispensable to create a tight link between classical natural product research, molecular medicine and organic chemistry. In the search for new lead structures, the selection of vegetable and animal organisms as well as fungi and microorganisms is performed according to the random principle, under chemotaxonomical aspects, on the basis of ecological observations and on the basis of ethnomedicinal previous knowledge.
Determining one or more active components from substance mixtures such as from substance libraries created by combinatorial chemistry or from natural product extracts, is, however, very labour-intensive.
Natural substance extracts, for example, generally consist of a large number (up to 2,000) of the most different substances spanning the entire polarity range, which is due to different basic structures and functional groups. As a rule, only relatively few compounds amount to already about 80% of the weight of the extract whereas the predominant part of the remaining compounds is present in low concentration down to the ppm region, i.e. non-equimolar. Frequently, however, only few substances, or even only one single substance, show the characteristic biological activity, and this activity may be caused by a substance which is present in the extract in traces.
Up to now, the processing and analysis of the mostly chromatographically separated components of a natural extract or of an extensive substance library generated by combinatorial chemistry has generally been performed using automated test systems with extremely high throughput (high-troughput screening; HTS). This method is, however, very labour- and cost-intensive. It is, for example, necessary to initially prepare from the natural product source (e.g. plant, animal, fungus, microorganism) selective extracts with solvents of increasing polarity and to subject these to biological tests. Further tests are made after subfractions have been formed from the respective effective selective extract.
Finally, an ultimate test is to show which pure substance(s), after isolation from the effective fraction, exhibit(s) biological activity and thus represents a “hit”. The chromatographical separation in sublibraries and the testing thereof require several weeks each. To be able to recover sufficient amounts of the pure substance(s), it is therefore necessary to start with large quantities of extract. This, too, entails high costs for preparative HPLC columns and the high solvent requirement (both purchase and disposal).
Already by separating the subfractions, but all the more so by isolating the pure natural substances, possible synergistic or antagonizing effects of the individual components of the extract are lost in high-throughput screening. Thus, an extract which is effective in the first test may lose its biological action because the separation into individual substances prevents target-binding, which target-binding was possible only by the interaction of various components.
A process for determining effective components from a synthetic peptide library created by combinatorial chemistry and consisting of maximally 19 chemically very similar peptides which originate only from the replacement of amino acids and are present in equimolar amounts, is described by Zuckermann et al., Proc. Natl. Acad. Sci. USA 89, 4505-4509 (1992). To this end, an antibody was added in deficient quantity to such a peptide substance library, and the target(=anti-body)-peptide complex was separated by rapid gel filtration. The peptide was set free from the complex with 1% trifluoroacetic acid, and the structure was elucidated by mass spectroscopy and amino acid analysis. This process is, however, unsuitable for target-molecule complexes of smaller molecules (molecular weight below or equal 1500) since gel filtration technically works only with greater differences in molecular weight. Also, according to the authors, the process requires equimolar mixtures. Furthermore, the determination of synergistically active combinations of ligands is impossible or left to chance.
The experiments described by Wieboldt et al. in Anal. Chem., 69, 1683-1691 (1997) are likewise directed to equimolar mixtures of 20 to 30, closely related molecules (synthetically produced derivatives having a general 1,4-benzodiazepine structure). The limited diversity of the synthetic substances does facilitate experimental processing, it is true, but at the same time represents a limiting factor for their use.
Likewise, the pulsed ultrafiltration mass spectrometry described by R. B. van Breemen et al. in Anal. Chem., 69, 2159-2164 (1997) requires an equimolar substance library with 20 substances. Since release is accomplished only with organic solvents, covalently bonded substances can not be detected.